SUMMARY (PROJECT 3) The overall scope of this project is to analyze CD4+ and CD8+ T cell responses specific for the four dengue virus serotypes (DENV1-4) observed in natural immunity and following vaccination and to define correlates of disease protection and susceptibility. To accomplish this goal, we have already identified CD8+ T cell epitopes for each of 27 common HLA A and B allelic variants in the general population from Managua, Nicaragua. Thus, we will now screen peripheral blood mononuclear cells (PBMCs) with HLA-matched peptide sets predicted to bind MHC class II molecules in these donors. The CD4+ and CD8+ epitopes identified will allow us to determine the balance of serotype-specific vs. conserved epitope responses in the general population and finally to test whether heterotypic infection is associated with differential quality of responses. To determine the role of CD4+ and CD8+ T cells in DENV protection or pathogenesis, we will then utilize the epitopes identified to test whether ?original antigenic sin? is associated with differential quantity and quality of responses in patients with documented clinical outcome and patterns of sequential infection. This will be accomplished by studying subjects associated either with defined infection outcome (inapparent vs. symptomatic), defined severity of disease (Dengue Fever, Dengue Hemorrhagic Fever, and Dengue Shock Syndrome), and subjects with defined patterns of sequential heterotypic DENV infection in samples distributed by Core C from P01- associated studies in Nicaragua (Aim 1). We further will test whether CD4+ and CD8+ T cell responses restricted by different HLA alleles are associated with differential magnitudes and frequencies in the general population and in patients with defined clinical outcome or inapparent infections (Aim 2). This will determine the rank of relative Odds Ratios of different HLA alleles as it relates to DENV-associated pathogenesis or protection. Finally, we will compare the magnitude, breadth and quality of CD4+ and CD8+ T cell responses elicited by vaccination with a tetravalent live attenuated dengue vaccine in DENV-nave and DENV-exposed populations. Samples from a Phase 3 efficacy trial will enable investigation of vaccine-induced correlates of protection (Aim 3). The results will be compared to correlates of protection identified under conditions of natural immunity. These studies are closely linked with other projects in this P01, as Project 1 will use the same samples/sample sets to investigate B cell and antibody correlates of protection from dengue disease and severe disease in natural DENV infections, and Project 2 will study B cell/antibody attributes of the same vaccinee samples. Overall, our studies will establish correlates of protection from DENV disease based on magnitude and multi-functionality of T cell responses, which will constitute an important contribution towards dengue vaccine development and evaluation.